The overall goal of this research is to elucidate the molecular mechanisms underlying neurotransmitter release in neurons. A considerable body of work over the last several years indicates that the docking and fusion of synaptic vesicles to the plasma membrane is mediated by a number of proteins including synaptobrevin (VAMP), syntaxin, and the 25 kDa synaptosomal- associated protein SNAP-25. In this proposal, the applicants focus on the role of SNAP-25 in neurotransmitter release. They hypothesize that the selective sorting of SNAP-25 and its function may be regulated by posttranslational modification and by its interaction with other synaptic proteins. They will attempt to provide direct evidence in support of their hypothesis using both in vitro and in vivo models. This research will lead to the understanding of how neurotransmitter release is controlled and it will ultimately enable us to design better treatment for neurological disorders caused by abnormal levels of neurotransmitters. The specific aims of this project are to: 1) Determine the site(s) and the dynamics of SNAP-25 palmitoylation/depalmitoylation. The finding that SNAP-25 is a major palmitoylated protein in the presynaptic terminals suggests that its functions may be mediated by dynamic acylation/deacylation. They will use site-directed mutagenesis to identify the amino acid residue(s) that is palmitoylated. They will also determine whether or not the palmitoylation of SNAP-25 is regulated during neurotransmitter release. 2) Determine if the intracellular sorting and recycling of SNAP-25 are regulated by palmitoylation and protein-protein interactions. The proper subcellular sorting and recycling of SNAP-25 are essential for its function in neurons. They will determine if palmitoylation and protein- protein interactions play a role in the subcellular localization and recycling of the protein in neurons. They will express several SNAP-25 mutants in PC12 cells and cultured rat embryonic neurons, and define the structural elements that are important for the sorting and recycling of SNAP-25. 3) Determine the role of SNAP-25 in neurotransmitter release. while recent studies suggested that SNAP-25 is required for neurotransmitter release, its precise role has not been defined. They will investigate the interactions between SNAP-25 and other synaptic proteins in the neuromuscular junction, and determine the significance of such interactions in regulating neurotransmitter release.